Method for treating nociceptive pain and burns with brazilian green propolis alcohol extract

ABSTRACT

A method for treating or preventing nociceptive pain by administering an ethanol extract of Brazilian green propolis and compositions comprising this extract.

BACKGROUND OF THE INVENTION Field of the Invention

The invention pertains to the fields of medicine, pain management, andapiculture.

Related Art

Pain is an uncomfortable feeling that involves motivational, cognitiveand affective aspects which serves as an alarm to minimize contact witha harmful agent. See Ruel, H. L.; Steagall, P. V. Adjuvant analgesics inacute pain management. VET. CLIN. SMALL ANIM. PRACT. 2019, 49,1127-1141.

Pain is initiated by nociceptors or specialized peripheral sensoryneurons which are found in muscle, cutaneous tissues, connectivetissues, vessels, viscera and bone; Zakaria, Z. A.; et al.Antinociceptive activity of petroleum ether fraction obtained frommethanolic extract of Clinacanthus nutans leaves involves the activationof opioid receptors and NO-mediated/cGMP-independent pathway. BMCCOMPLEMENT. ALTERN. MED. 2019, 19, 79. These nociceptors are activatedby noxious stimuli including by chemical, mechanical and thermal stimulithat trigger appropriate response to tissue damage or injury. However,when pain persists, it affects the quality of life of a person whosuffers from it; Goucke, C. R. & Chaudakshetrin, P. Pain: A neglectedproblem in the low-resource setting. ANESTH. ANALG. 2018, 126,1283-1286.

Conventional pain management techniques include the administration ofanalgesics and anti-inflammatory agents; De Moraes, S. Z.; et al.Antinociceptive and anti-inflammatory effect of Poincianella pyramidalis(Tul). LP QUEIROZ. J. ETHNOPHARMACOL. 2020, 254, 112563. However, theseagents often exhibit detrimental side-effects. For example,non-steroidal anti-inflammatory drugs (NSAIDS) can induce severe orlife-threatening gastrointestinal (GI) bleeding, cause ulcers orincrease the risk of heart attacks or strokes. NSAIDS also can produceother undesirable or toxic side effects on the gastric system (stomachand intestines), liver and kidneys.

Due to adverse effects or toxicities associated with conventional paintherapy, there is a need for the identification and development of newand effective agents with no or fewer undesirable side effects; De SouzaSampaio, R.; et al., Antinociceptive activity of the Psidium brownianumMart ex DC. leaf essential oil in mice. FOOD CHEM. TOXICOL. 2020, 135,111053, incorporated herein by reference in its entirety.

Natural products have been pursued as possible alternatives toconventional analgesics; Islam, N. U. et al., Antinociceptive, musclerelaxant and sedative activities of gold nanoparticles generated bymethanolic extract of Euphorbia milli. BMC COMPLEMENT. ALTERN. MED.2015, 15, 1-11. Such natural products are often readily available inareas that are underserved by conventional pharmaceutical medicines.

In the last three decades, led by the World Health Organization (WHO),interest in natural products has greatly increased. The WHO recentlypublished a document entitled WHO traditional medicine strategy:2014-2023, in which it endorsed and supported the development andimplementation of action plans to strengthen the role traditionalmedicines in public health; World Health Organization. WHO TRADITIONALMEDICINE STRATEGY: 2014-2023; World Health Organization: Geneva,Switzerland, 2013.

Different chemical compounds, such as particular flavonoid orphytochemicals, having potent analgesic effects have been obtained fromnatural sources. Such natural analgesics have been explored as possiblelead compounds for the development of new analgesic drugs; Abdel-Rahman,R. F. Natural products as a source for novel analgesic compounds. PAINRELIEF ANALG. ALTERN. THER. INTECH OPEN 2017, 277-299.

Propolis (bee glue) is a natural resinous mixture produced by honey beeswhich is produced when bee-released compounds are combined with exudatesgathered from various plant sources; Al-Hariri, M. T. Propolis and itsdirect and indirect hypoglycemic effect. J. FAM. COMMUNITY MED. 2011,18, 152. Propolis may be fractionated by various means including byproduction of propolis extracts in different solvents such as water,ethanol or oil.

As disclosed herein, the inventors investigated and explored potentialanalgesic properties of propolis on nociceptive pain. As disclosedherein, the inventors describe the identification, formulation and useof different fractions or extracts of Brazilian green propolis as agentsto ameliorate nociceptive pain or exhibit other analgesic properties.

SUMMARY OF THE INVENTION

One aspect of the disclosure is a method for preventing or treatingnociceptive pain comprising, consisting essentially of, or consistingof, applying an ethanol extract of Brazilian green propolis (“BGP”) to asite at risk of thermal injury or to a burn. In some embodiments thismethod further comprises applying a mixture of both an ethanol and anoil extract of BGP to a site at risk of thermal injury or to a burn.

Another aspect of the invention is directed to a composition comprising,consisting essentially of, or consisting of an ethanol extract of BGP ora material derived from such an extract in a form suitable forapplication to a site at risk of thermal injury or to a burn. Thiscomposition may further comprise an oil extract of BGP or a materialobtained from such an oil extract. The composition may also containother active ingredients such as NSAIDs or opioid analgesics or beprepared as part of a wound or burn dressing.

One aspect of the invention is a method for preventing or treating pain,or more specifically, nociceptive pain from a burn or thermal injurycomprising administering to a subject in need thereof a compositioncomprising an ethanol extract of Brazilian green propolis (“BGP”).Preferably, the method is directed to treating non-inflammatory pain orearly phase responses mediated by C-fiber activation.

The organization of rodent dorsal horn laminae is largely similar tothat in humans which is why animal model is similar to human; seeKaliyaperumal, S. et al., TOXICOLOGICAL PATHOLOGY, 2019, 48, 202-219;Zotova, E. G. et al., PHYSIOLOGY JOURNAL, 2013, Article ID 254789; andSerra, J. et al., PAIN, 2012, 153(1)m 42-55 each incorporated byreference.

Nerve endings of nociceptive C fibers (nociceptors) are capable totranslate an aggressive thermic, chemical, or mechanical stimulus intoan electrical stimulus that will be transmitted to the central nervoussystem and interpreted in the cerebral cortex as pain. However, in theintact animal and in humans, nociceptive input reaches subcortical andcortical brain nuclei that contribute to the affective, aversive statesof pain. Hypertext transfer protocolsecure://www.ncbi.nlm.nih.gov/books/NBK32655/.

In another embodiment, the pain or nociceptive pain further compriseslate phase response resulting from an inflammatory response incombination with activation of N-methyl-d-aspartate (NMDA) and non-NMDAreceptors and NO cascade).

Over the past few years, a number of animal models have been developedthat, to a large extent, mimic the nociception originating in theviscera; Larauche, M., et al., EXP. NEUROL. (in press); Al-Chaer, E. D.et al., PAIN, 2002, 96(3), 221-225. These models have helped advance ourunderstanding of the acute physiological responses associated withmechanical or inflammatory visceral nociception. It has become apparentthat visceral pain and somatic pain are different, although somesimilarities exist; Al-Chaer et al, supra. In the present study, usedthe acetic acid-induced nociceptive model was developed for thescreening of analgesic drugs and described as the writhing test to examthe anti-nociceptive effect of minocycline in acute visceral pain. I.p.injection of acetic acid is a well-known noxious chemical visceralstimulus in animals; Koster, R, et al., FED PROC, 1959, 18, 412. Thenociceptive stimulation of the peritoneum by i.p. acetic acid producesabdominal contractions or writhes, and gastrointestinal ileuscharacterized by inhibition of gastric emptying and small intestinetransit. Both procedures are associated with visceral pain. This modelis also used as a model of somatic-visceral pain; Ghia, et al., LIFESCI, 2004, 75, 1787-1799.

In preferred embodiments of this method, the BGP ethanol extract isprepared by extracting BGP with an aqueous solution containing 5 to 100vol. % of ethanol, preferably from 60-80 vol. % ethanol.

In some embodiments, the composition further composition furthercomprises an oil extract of BGP in combination with an ethanol extract.In an alternative embodiment, the method may be practiced with acomposition comprising an oil extract of BGP.

In some embodiments the composition further comprises, or comprises, avegetable oil extract of BGP.

In other embodiments, the composition used in the method disclosedherein comprises extract of BGP that has been emulsified with theethanol extract and/or oil extract of BGP.

In further embodiments, the disclosed method may use a compositionfurther comprises a NSAID, an opioid, or other analgesic or anesthetic.

In other embodiments, the composition used in the disclosed methodfurther comprises a sunblock or sunscreen and is topically administeredto the subject. In one embodiment, the composition comprises a hydrogel.

In one embodiment, the composition comprises an emulsion or liposomeswhich may comprise an oily and/or aqueous phase containing the ethanoland/or oil extract of BGP.

In another embodiment, the composition comprises a sunscreen orsunblock.

Another aspect of the disclosure is directed to a burn dressingcomprising an ethanol and/or oil extract of BGP.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a bar graph illustrating the antinociceptive effect ofBrazilian green propolis alcohol extract (P50 and P100) in the infraredtest in the rats. The values expressed are mean±SEM, (n=6).* p<0.05:significantly different for treated (P50 and P100) compared to thecontrol groups as determined by ANOVA analysis followed by multiplecomparison post hoc tests.

FIG. 2 is a bar graph illustrating the effect of Brazilian greenpropolis alcohol extract (P50 and P100) in the formalin-inducednociception model: neurogenic (Phase I) and inflammatory (Phase II). Thevalues expressed are mean±SEM, (n=6). * p<0.05: significantly differentfor treated (P50 and P100) compared to the control groups as determinedby ANOVA analysis followed by multiple comparison post hoc test; and

FIG. 3 is a bar graph illustrating the effect of Brazilian greenpropolis alcohol extract (P50 and P100) against acetic acid-inducedabdominal contraction in rats. The values expressed are mean±SEM (n=6).*p<0.05: significantly different for treated (P50 and P100) compared tothe control groups as determined by ANOVA analysis followed by multiplecomparison post.

FIG. 4A describes certain chemical structures of components of Braziliangreen propolis.

FIG. 4B describes the chemical structure of Artepillin C.

FIG. 5 describes the chemical structures of certain volatile componentsof Brazilian green propolis.

FIG. 6 describes the differences in the chromatographic chemicalfingerprints of red and green propolis.

FIG. 7 describes nociceptive pain fibers and pathways.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As explained above, new treatment strategies and therapeutics fortreating nociceptive pain are needed because conventional painmanagement techniques and pharmaceuticals do not adequately treatdifferent types of nociceptive pain and/or have undesirableside-effects. With this objective in mind, the inventors sought toidentify new treatment strategies for nociceptive pain management usingnatural materials derived from Brazilian green propolis (BGP).Specifically, as disclosed herein, the properties of ethanol extracts ofBrazilian green propolis were evaluated using a variety of in vivomodels of different types of nociceptive and inflammatory pain. Amongother findings, the results demonstrated that BGP ethanol extractssignificantly reduced visceral nociceptive pain, increased the painthreshold against pain induced by infrared radiation, and inhibitedchemically (formalin) induced pain. Administration of BGP extractreduced both non-inflammatory nociceptive pain as well as some types ofinflammatory nociceptive pain.

The BGP compositions used in the presently disclosed method may beadministered in a form suitable for ameliorating or reducing theseverity of nociceptive pain. In some embodiments, the composition istopically administered to the subject; in other embodiments thecomposition is administered to the subject prior to a burn; in anotherembodiment the composition is administered to a subject at risk of beingburned and who handles or works around hot liquids, hot surfaces, orflames.

Another aspect of the disclosure is directed to a formulation comprisingan ethanol extract and/or oil extract of BGP and an excipient suitablefor application to the skin or to a burn. Such a composition preferablycomprises a gel or spray which may further comprise other activeingredients such as s an NSAID, opioid, analgesic or anesthetic.

Brazilian Green Propolis.

Propolis is formed by a complex set of components collected by Apismellifera bees from different parts of plant resins (e.g., twigs,flowers, pollen, buttons, and exudates of trees) which are deposited inthe hive with saliva and enzymes of the insect to seal cracks andmaintain the temperature in the hive. Standardized Brazilian greenpropolis products are commercially available, such as those sold by ApisFlora Company (hypertext protocol secure://www.apisflora.com.br/cataloglast accessed Apr. 14, 2021). Green propolis may be harvested from hivesin the Brazilian states or regions of Minas Gerais (southeast) or Parana(south) or other states or regions of Brazil.

Propolis comprises resins with about 40-60 wt % of the mixture beingcomposed of flavonoids and phenolic acids usually about 50 wt %; about25-35 wt % waxes, usually about 30 wt %; about 5-15 wt % essential oilsusually about 10 wt %; and about 1-10 wt % pollen, usually about 5 wt %.Propolis can be composed of hydrocarbons, alcohols, aliphatic andaromatic acids, esters and its derivatives, aldehydes, ketones,flavonoids, fatty acids, terpenoids, amino acids, sugars, lignans,vitamins, minerals, and other components.

An ethanol extract may contain various concentrations of flavonoidclass, prenylated derivatives of p-coumaric acid. coumaric and ferulicacid, pinobanksin, isosakuretin and kaempferide, isopentenylatedphenylpropanoids and their derivatives, caffeoylquinic acids, someterpenes, phenolic acids and has an especially high level of ArtepillinC (3,5-diisopentenyl-4-hydroxycinnamic acid); as described by andincorporated by reference to Simoes, L. M. C. et al., J.ETHNOPHARMACOLOGY, 2004, 94(1), 59-65.

The chemical composition of propolis differs significantly according tothe geographic region where resins were collected due to the flora ofeach region, allowing the selection of different plants as source ofresin. When this product is derived from Europe or China, for example,the main plant metabolites found are flavonoids and phenolic acids,unlike BGP s from southeastern Brazil, which, besides phenoliccompounds, contain high amounts of terpenoids and prenylated derivativesof p-coumaric acid such as those shown by FIGS. 4A and 4B.

These differences in composition reveal the collection of resinousmaterial in temperate zones from poplar, especially species of Populus;and in southeastern Brazil from Baccharis dracunculifolia DC(Compositae), Baccharis dracunculifolia DC (Asteraceae) is the mainbotanical source used by honeybees to produce Brazilian green propolis.

Brazilian green propolis is composed of large amounts of phenoliccompounds such as Artepillin C, baccharin, kaempferide, isosakuranetin,dihydrokaempferide, drupanin, p-coumaric acid, caffeic acid,aromadendrin, caffeoylquinic acid derivatives, and other compounds, suchas the triterpene lupeol-3-(3′R-hydroxy)-hexadecanoate. The key sourceof these compounds is B. dracunculifolia.

FIG. 4A describes the chemical structures of compounds found inBrazilian green propolis and FIG. 4B describes the chemical structure ofArtepillin C a component of Brazilian green propolis. The major volatilecomponents of Brazilian green propolis are sesquiterpenes, such as(E)—nerolidol, β-caryophyllene, spathulenol, and δ-cadinene; FIG. 5 .Furthermore, other compounds such as selina-3,7(11)diene,benzenepropanoic acid and longipinene were also identified. According toFernandes-Silva, C. C., et al., J. Sci. Food Agric, 2015, 95, 309105,the main constituents of a volatile fraction of BGP are thephenylpropanoid 3-prenylcinnamic acid allyl ester (26.3%) and thesesquiterpene spathulenol (23.4%). It also contains several othersesquiterpenes and phenylpropanoids, in addition to linalool andα-terpineol (monoterpenes). Such volatile components may be extractedinto water, ethanol or other alcohol, oil, or other solvent.

As shown by FIG. 6 , Brazilian green propolis contains little or noformononetin but contains significant amounts of drupanin, Artepillin Cand bacharin compared to red propolis. See Berretta, A. A., et al.,Functional Properties of Brazilian Propolis: From Chemical CompositionUntil the Market, 2017, DOI: 10.5772/65932. Available from: hypertexttransfer protocolsecure://www.intechopen.com/books/superfood-and-functional-food-an-overview-of-their-processing-and-utilization/functional-properties-of-brazilian-propolis-from-chemical-composition-until-the-market(last accessed Apr. 2, 2021, incorporated by reference for itsdescription of the chemical composition of, functional properties of,distinguishing features of green propolis compared to other types ofpropolis, such as red or brown propolis, sources of, production of, andother description of Brazilian propolis. Those skilled in the art candetermine the content ranges of these and other components of BGP incommercially available BGP, such as those available from the Apis FloraCo.

The concentrations (wt/vol or wt/wt) of each these components, includingArtepillin C, in a water, ethanol, or oil extract as described herein,may be <10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400or >400% more than those found in raw BGP.

Brazilian Green Propolis Water Extract.

A water extract as described herein includes those made using an aqueoussolution, preferably a solution with no or substantially no ethanol. Anaqueous solution may contain salts, buffers, acids or bases or otherwater-soluble components. It may be produced in a manner similar to thatdescribed below for an ethanol extract.

Brazilian Green Propolis Ethanol Extract.

An ethanol extract is produced by extracting BGP in a solvent containingethanol. BGP alcohol extracts may be produced by extracting BGP in otheralcohols or aqueous alcohols, such as those containing methanol,propanols, butanols, or combinations of alcohols. Water or an aqueousbuffer may be present. Methods for producing extracts of BGP, includingethanol extracts, are described by and incorporated by reference toDevequi-Nunes D, et al., Chemical characterization and biologicalactivity of six different extracts of propolis through conventionalmethods and supercritical extraction. PLoS ONE, 2018, 13(12): e0207676.To prepare an ethanol extract, BGP may be processed by chopping,grinding, milling, powdering or otherwise disintegrating prior toextraction to provide a larger surface area and smaller particle volumefor extraction. An extract may be produced by dissolving solublecomponents of BGP in a mixture of water and alcohol.

Ethanol is a very polar molecule due to its hydroxyl (OH) group, withthe high electronegativity of oxygen allowing hydrogen bonding to takeplace with other molecules. Ethanol therefore attracts polar and ionicmolecules. The ethyl (C₂H₅) group in ethanol is non-polar. Ethanoltherefore attracts non-polar molecules. The inventors considered that anethanol extraction might separate or concentrate ethanol-solublecomponents of BGP that exhibit analgesic effects, especially on earlyphase nociceptive pain, from other inactive or pain-inducing componentsof BGP.

A concentration of ethanol suitable for extracting analgesic compoundsfrom BGP may be selected by one skilled in the art and such extractsassessed for activity as disclosed herein. Such a mixture may compriseabout <5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or >95% (vol/vol)ethanol with the balance being water or an aqueous solution or buffersuch as normal saline, phosphate-buffered saline (PBS), Tris buffer, orother aqueous solution, preferably a physiologically acceptablesolution. Preferably, a concentration of ethanol ranging from 40 to 70%or 50, 55 to 60% (vol/vol) is employed.

The extraction may be performed at a pH ranging from <4, 4, 5, 6, 7, 8,9, 10, or >10, preferable at a pH from about 6, 6.5, 7, 7.5 to 8. The pHmay be adjusted by known methods, such as by addition of, or titrationwith, an acid, such as HCl, or a base, such as NaOH. The pH may also beadjusted or stabilized by addition of a buffer, such as Tris. Bufferswith particular pH buffering ranges, including MES, ACES, PIPES, HEPES,Tricine, Gly-Gly, CAPS, CABS, and others are commercially available andincorporated by reference to hypertext transfer protocolsecure://www.sigmaaldrich.con/life-science/core-bioreagents/biological-buffers/leaming-center/buffer-reference-center.html#selectedbuffers.

An ethanol extract of BGP may include phenolic compounds, flavonoids,and antioxidants. Representative compounds include Artepillin C,2,2-diphenyl-1-picrylhydrazyl (DPPH), p-coumaric acid, and2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The extractsdisclosed herein may be produced or stored under an inert atmosphere,such as nitrogen or a noble gas, or in the absence of oxygen. Extractsmay be stored at a temperature <0, 4, 5, 10, 20, 25 or >25° C.,preferably below room temperature.

Standardization of an ethanolic extract of BGP may be based on thecontent of these or other compounds as they appear in raw propolis, itsvolatile components, or its fractions. For example, a standardizedextract may contain a reference amount of Artepillin C or phenolcompounds or flavinoids.

Ethanol extracts of BGP contain a high content of flavonoids andArtepillin C as described by and incorporated by reference toShahinozzaman, M. FITOTERAPIA, 2020, 147, 104775. These compounds may beassociated with analgesic activity.

Ethanol extracts of Brazilian green propolis are commercially available(e.g., from Uniflora, hypertext protocol secure://www.uniflora.us/, lastaccessed Apr. 21, 2021, incorporated by reference). Uniflora catalognumbers include Item #19150 (BRAZILIAN GREEN BEE PROPOLIS LIQUID EXTRACTALCOHOL FREE 60 BRIX), Item #19125 (BRAZILIAN GREEN BEE PROPOLIS LIQUIDEXTRACT 35%); Item #191130 (BRAZILIAN GREEN BEE PROPOLIS LIQUID EXTRACTMEDIUM TYPE 45%); and Item #19140 (BRAZILIAN GREEN BEE PROPOLIS LIQUIDEXTRACT STRONG TYPE 55%). These commercial extracts contain componentsextracted from BGP using ethanol.

Commercially available ethanol extracts of BGP contain caffeic acid,p-coumaric acid, trans-cinnamic acid, aromadendrin and Artepillin C. Insome embodiments, one or more of these components of an ethanol extractmay be used alone or in mixture with other such components or incombination with ethanol extract of BGP (fortification), in a method asdisclosed herein.

In some embodiments, an ethanol or water extract of BGP is desiccated orfreeze dried to remove solvents to produce a solid material. This solidmaterial may be resuspended in a solute of choice, preferably aphysiological solute such as normal saline or PBS for administration. Inother modes the solid material may be administered as a solid, forexample, in a capsule or tablet or as a troche. An ethanol, water, oroil extract may be further processed to extract particular components ofthe extract.

In some alternate embodiments, other alcohols, such as methanol,propanol or pentanol or hexanol may be used to produce an alcoholicextract of BPG. Ethanol is considered a universal solvent, as itsmolecular structure allows for the dissolving of both polar, hydrophilicand nonpolar, hydrophobic compounds. As ethanol also has a low boilingpoint, it is easy to remove from a solution that has been used todissolve other compounds, making it a popular extracting agent forbotanical oils. In ethanol the C₂H₅ moiety is hydrophobic, but the OHgroup is polar giving rise to a dipole moment. The OH group can act as ahydrogen bond donor and a hydrogen bond acceptor with water. As thelength of the alkyl chain increases, the hydrophobic effect increases,thus while methanol, ethanol, propanol are miscible with water, higheralcohols such as butanol (0.11 alcohol solubility in water), pentanol(0.030), hexanol (0058), and heptanol (0.0008) are much less so andafter heptanol, the higher alcohols are considered immiscible.

Similar proportions of water and alcohol to those described above may beused during extraction. Preferably a water miscible alcohol, such asmethanol or propanol is selected, however, in some embodiments waterimmiscible alcohols (or other immiscible solvents) may be used toextract BGP and extract materials recovered from either a water phase oralcohol phase. BGP may also be extracted using supercritical carbondioxide. Sources of BGP and extraction methods including using ethanolor supercritical carbon dioxide are described by and incorporated byreference to Machado, et al., hypertext transfer protocolsecure://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706314 2016; 11(1):e0145954. As explained below, BGP may also be extracted using non-polaror hydrophobic solvents.

Surprisingly, in view of the presence of Artepillin C, the inventorsfound that an ethanol extract of BGP inhibits nociceptive pain becauseethanol extracts. Artepillin C (FIG. 4B) is a compound that activatesTRPA1 which is an ion channel sensor for pain, cold and itch; see Hata,T. et al., Artepillin C. a major ingredient of Brazilian propolis.induces a pungent taste by activating TRPA1 channels, PLOS ONE.2012.7(11):e48072. doi: 10.1371/journal.pone.0048072. Epub 2012 Nov. 2.TRPA1 is an ion channel located on the plasma membrane of many human andanimal cells. This ion channel is best known as a sensor for pain, coldand itch in humans and other mammals, as well as a sensor forenvironmental irritants giving rise to other protective responses(tears, airway resistance, and cough); see Andersen H H, et al. HumanSurrogate Models of Histaminergic and Non-histaminergic Itch, ACTADERMATO-VENEREOLOGICA. 2015. 95 (7): 771-7. doi:10.2340/00015555-2146.PMID 26015312; and Hojland C R, et al., A human surrogate model of itchutilizing the TRPA1 agonist trans-cinnamaldehyde. ACTADERLMATO-VENEREOLOGICA. 2015, 95 (7): 798-803.doi:10.2340/0001SSSS-2103. PMID 25792226.

While not being bound to any theory or explanation. the inventorsbelieve that components in an ethanol extract such as Artepillin C orother flavonoid compounds such as pinobanksin and kaempferol and thephenolic acides3,5-diprenyl-4-hydroxcinnamic acid,2,2-dimethyl-6-carboxyethenyl-8-prenyle-2H-1-benzopyran and3-prenyl-4-hydroxycinnamic may produce analgesic effects via inhibitionof inducible NOS (iNOS) activity.

Brazilian Green Propolis Oil Extract:

The Brazilian green propolis oil extract as used herein typicallycomprises a vegetable oil extract of Brazilian green propolis. However,in some embodiments mineral oils or other hydrophobic or non-ionicsolvents may be used. In a preferred embodiment, the Brazilian greenpropolis extract comprises canola oil. An oil extract of GPB may containHydroxy-3-prenylcinnamic acid, 3,4-Dihydroxy-5-prenylcinnamic acidIsosakuranetin, Kaempferide, Dihydrokaempferide, and(E)-3-{-4-hydroxy-3-[(E)-4-(2,3-dihydrocinnamoyloxy)-3-methyl-2-butenyl]-5-prenylphenyl}-2-propenoicacid as well as Artepillin C. These and other components of GPB oilextract and their properties are described by, and incorporated byreference to see Cavalho, et al., In vivo antitumoural activity andcomposition of an oil extract of Brazilian propolis, FOOD CHEMISTRY,2011, 126(3), 1239-1245. and Shahinozzaman, et al., Artepillin C: Acomprehensive review of its chemistry, bioavailability, andpharmacological properties, F ITOTERAPIA, 2020, 147, 104775.

The components of Brazilian green propolis that are extracted into theoil (or water or ethanol) may be further isolated, for example, bychromatography or phase-partitioning. One example is partitioning ofBrazilian green propolis oil in an alcohol or aqueous mixture of analcohol substantially immiscible with the oil, and recovery of thefraction of propolis compounds partitioning into the aqueous alcohol.The isolated fraction of Brazilian green propolis may be used by itself,resuspended in oil, or admixed with other carriers or excipients.

In some embodiments, the Brazilian green propolis oil extract or ethanolextract comprises 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 to 5 wt % ofphenolic acids or other phenolic compounds. This range, and other rangesdisclosed herein, include all intermediate and terminal values as wellas subranges.

In other embodiment the Brazilian green propolis oil extract or ethanolextract comprises 00.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 to 5 wt %of flavonoids.

In one embodiment, the Brazilian propolis oil extract or ethanol extractcomprises 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 to 5 wt % ofArtepillin C.

Other Extracts of BGP.

In some embodiments, other extracts of BGP may be used, for example,those extracted in hydroxy-3-prenylcinnamic acid,3,4-dihydroxy-5-prenylcinnamic acid, isosakuranetin, kaempferide,dihydrokaempferide, and(E)-3-{-4-hydroxy-3-[(E)-4-(2,3-dihydrocinnamoyloxy)-3-methyl-2-butenyl]-5-prenylphenyl}-2-propenoicacid or those described by and incorporated by reference to Carvalho, etal., FOOD CHEMISTRY, 2011, 126(3), 1239-1245.

Brazilian green propolis, its oil extract or oil extract fractions (orits water or ethanol extracts), in some embodiments may be characterizedor standardized based on a the phenylpropanoid 3-prenylcinnamic acidallyl ester ranging from about 23-29 wt %, preferably about 26.3%, and20 to 20 wt % sesquiterpene spathulenol preferably about 23.4 wt %.Standardization may be based on the content of these or other compoundssuch as Drupanin, Artepillin C and Baccharin as they appear in rawpropolis, its volatile components, or its fractions, such as its oilfraction, ethanol fractions or aqueous fractions.

Compositions Comprising More than One BGP Extract.

In some embodiments, a mixture of water-ethanol, ethanol-oil, orwater-oil extracts may be formulated, for example, at a ratio rangingfrom <12:1, 12:1, 10:1, 8:1, 6:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:6, 1:8,1:10, or 1:12 or >1:12 (or any intermediate ratio or subrange ofratios). In another embodiment, such a mixture may contain two or threedifferent propolis extracts, wherein each extract is present in anamount ranging from <5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or >95vol. %. Preferably, a combination of two or three different Braziliangreen propolis extracts will produce a synergistic or additiveanti-nociceptive effect on nociceptive, noninflammatory, or burn pain.

Inactive Components.

A BPG extract composition may contain other ingredients besides a BGPextract such as one or more carriers or excipients, for example, it maycontain one or more emulsifiers or excipients, pH buffers, salts,preservatives, or antioxidants. The composition may be substantiallyhydrophobic, a mixture of hydrophobic and aqueous or polar components,or substantially aqueous or polar. Compositions include those containingmixtures of oils, water and water-miscible solvents, W/O or O/Wemulsions, ointments or salves, pastes, liposomes, gels, foams, films,or varnishes may contain components of an ethanol and/or oil extract ofBGP.

Compositions containing Brazilian green propolis ethanol extracts or BGPoil extract may include solvents (e.g., aqueous solvents), buffers,emulsifiers, surfactants, semisolid or solid carriers, film-formingingredients, propellants, or other excipients Emulsifiers include, butare not limited to, Agar, Albumin, Alginates, Casein, Ceatyl Alcohol,Cholic acid, Desoxycholic acid, Diacetyl tartaric acid esters, lecithin,and glycerol. Surfactants include, but are not limited to, ethoxylates,ethylene oxide/propylene oxide (EO/PO) copolymers, fatty alcoholethoxylates, alkylphenol ethoxylates, and fatty acid ethoxylates.Preferably an emulsifier or surfactant commonly used in a skincareproduct is used.

Active components. A Brazilian green propolis extract composition maycontain other active ingredients, such as analgesics includingnon-steroidal anti-inflammatory drugs (NSAIDs) such as salicylates andibuprofen. Other analgesics may be incorporated including opioidreceptor modulators, sodium channel blockers, calcium channel blockersTRP channel modulators, cannaboid receptor modulators, nerve growthfactor inhibitors. Examples of these analgesics are incorporated byreference to hypertext transfer protocolsecure://en.wikipedia.org/wiki/List_of_investigational_analgesics (lastaccessed Apr. 15, 2021). Preferably, a combination of Brazilian greenpropolis extracts with another active ingredient will produce asynergistic or additive effect on nociceptive, noninflammatory, or burnpain.

Skincare.

A Brazilian green propolis extract composition may be incorporated intoa skin protectant or skin restoring product, such as an ultravioletprotectant, sunscreen, sunblock or post-UV exposure protectant. Theseproducts typically contain one or more occlusive agents which create abarrier that blocks water from escaping the skin, such as petrolatum,mineral oil and/or dimethicone; one or more humectants, which areingredients that attract water, such as glycerin or glycerates; and oneor more emollients, such as coconut oil, cetyl esters, and silicones;which improve the feel of a protectant on the skin and reduce thetackiness and greasiness caused by the other moisturizing ingredients.

Sunscreen.

In some embodiments at least one compound that screens out, filters, orblocks UV radiation may also be incorporated. Such compounds includepara-aminobenzoic acid (PABA), padimate O, phenylbenzimidazole sulfonicacid, cinoxate, dioxybenzone, oxybenzone, homosalate, menthylanthranilate, octocrylene, octyl methoxycinnamate, octyl salicylate,sulisobenzone, trolamine salicylate, avobenzone, ecansule, titaniumoxide, zinc oxide and mixtures thereof. In contrast to conventionalsunscreens or sunblocks, the composition of the invention contains aBrazilian green propolis extract which further protects againstnociceptive paid caused by exposure to harsh conditions, such as todirectly sunlight on a beach.

In one embodiment, an ethanol and/or oil extract containing compositionmay be applied to a burn to prevent further aggravation of the burn ornociceptive pain or itching from the burn for example, which is causedby subsequent exposure to UV or infrared light which induces pain orcauses additional inflammation to a prior burn. The application of a BGPcomposition as disclosed herein to an existing burn can block newafferent pain signaling caused by subsequent aggravation of an existingburn by UV or infrared exposure, for example, by blocking new painsignaling transmitted via C fibers and other afferent myelinated fibers.

A composition comprising an ethanol and/oil extract as disclosed hereinmay be used in conjunction with laser or photodynamic therapy, forexample, by its application to skin or tissue before, during or aftersuch a therapy to avoid or mitigate pain.

In another embodiment of the present disclosure an individual sufferingfrom a skin burn such as a first-degree or second-degree burn can besubject to treatment with light to further reduce pain caused by theburn. In this embodiment of the invention the skin area damaged by theburn e.g., excessive exposure to sunlight and/or heat in amountssufficient to cause a first or second degree burn, and exhibitingcharacteristic pain symptoms, is first treated with exposure to UV lightprior to application of a BGP composition to the burn area. Theapplication of UV light is carried out immediately prior to theapplication of the BGP composition, preferably within 30 minutes, 20minutes, 10 minutes or 5 minutes prior to application of BGPcomposition. The application of UV light to a skin surface suffering aprior burn may lead to a minor aggravation or sensitization of theexisting burn. However, the subsequent application of a BGP compositionis especially effective for providing quick pain relief and almostimmediately after application of the BGP composition, e.g., a patienttreated in this manner may report no pain symptoms within 30 minutes,preferably 10 minutes or 5 minutes of application of the BGPcomposition. Preferably the UV light used to expose the burned skinsurface is mainly within a wavelength of 280-400 nm, preferably 320-400nm, which is typically regarded as including the UVA and UVB portions ofthe light spectrum. The effect of quick reduction in discomfort frompain is not as prevalent for treatment using secondary exposure to UVClight wavelengths.

The UV exposure is preferably of greater intensity for short periods oftime. Preferably the UV exposure occurs briefly (over a period of timeof 10 minutes or less, preferably five minutes or less) at levels of1-50 milliwatt per square centimeter, preferably 10-40 mW/cm² or about25 mW/cm² of direct exposure to UV light from a bulb that preferentiallyemits light in the UVB portion of the light spectrum.

Natural Ingredients.

A composition containing Brazilian green propolis water, ethanol or BGPoil extracts include essential oils such as eucalyptus, cedarwood,clove, Litsea cubeba, Mentha arvensis, rosemary, rose hips, garlic,sweet organe, sweet basil, melissa, cajeput, chamomile, wintergreen,turmeric, peppermint, menthol, camphor or lavendar oils; tea tree oil,hemp oil, coconut oil, palm oils, medium chain triglycerides, sheabutter, jojoba oil, cocoa butter; or capsicum extracts such ascapsaicin, dihydrocapsaicin, nordihydrocapsaicin, capsaicinoids,curcuminoids, gingerols, shogaols, zingerones, gingerdiols, orgingerdione. Other essential oils are described by and incorporated byreference to hypertext transfer protocolsecure://en.wikipedia.org/wiki/Essential_oil (last accessed Apr. 15,2021). Alternatively, other oily or hydrophobic ingredients such aspetrolatum or mineral oil may be used in a composition comprising a BGPethanol or oil extract.

Vitamins.

The Brazilian green propolis water, ethanol, or oil extracts may beadmixed with fat soluble vitamins such as vitamin A (retinoic acid,other retinoids, carotenes), D, E, K1 (phylloquinone) or K2(menaquinone). Vitamin A functions to keep skin and mucous membraneshealth and promotes growth. Vitamin D is involved in bone and teethhealth and strength. Vitamin is a fat soluble antioxidant which canprotect cell membranes. Vitamin K is involved in blood clotting,maintaining bone health and preventing calcification of arterial andother tissues. These vitamins in combination with Brazilian greenpropolis water, ethanol or oil promote additive, complementary, orsynergistic analgesic and/or regenerative effects. Emulsifiers orsurfactants, such as those described below, may be included tofacilitate admixture of hydrophobic and hydrophilic extracts or theircomponents.

The Brazilian green propolis ethanolic extracts or BGP oil may also beadmixed with water soluble vitamins such as the B vitamins like B1(thiamine), B2 (riboflavin), B3 (niacin), B4 (adenine), B5 (panthothenicacid), B6 (pyroxidine), B7 (biotin), B8 (inositol), B9 (folate) and B12,or vitamin C (ascorbic acid, ascorbyl palmitate). Gamma-aminobutyricacid may be included in some combinations. Vitamins B1, B6, B12 and GABAin addition to their effects on nociceptive or non-inflammatorynociceptive pain may reduce neuropathic pain. Emulsifiers or surfactantsmay be included to facilitate admixture of hydrophilic and hydrophobicextracts or their components.

Co administration of the vitamins or nutrients described above canreduce or eliminate nociceptive pain or non-inflammatory nociceptivepain as well as promote healing of damaged skin, mucous membranes orother distressed tissues. Such methods and compositions may be appliedto treatment of neuropathy, such as diabetic neuropathy, and chronicpain.

In some embodiments, the Brazilian green propolis ethanol extracts orBGP oil extracts incorporate fluids used in medicine or dentistry suchas saline solutions, rinses, lavage fluids, dialysis fluids, orartificial saliva. Brazilian green propolis ethanol extracts or BGP oilextracts may be incorporated into a microparticle or nanoparticle; agel, foam, or spray; an emulsion, liposome, ointment, paste, salve; or afilm.

Routes of Administration.

The methods of administering the BGP extracts or compositions containingthem disclosed herein may comprise administering a silver or titaniumoxide nanoparticle composition or composition intravenously,intramuscularly, topically (e.g. on or into a wound or lesion),intradermally, into or over a wound, intramucosally, subcutaneously,sublingually, orally, intravaginally, intracavemously, intraocularly,intranasally, intrarectally, gastrointestinally, intraductally,intrathecally, subdurally, extradurally, intraventricular,intrapulmonary, into an abscess, intra articularly, into a bursa,subpericardially, into an axilla, intrauterine, into the pleural space,intraperitoneally, transmucosally, or transdermally.

Carriers/Excipients.

The term carrier encompasses any excipient, binder, diluent, filler,salt, buffer, solubilizer, lipid, stabilizer, or other material wellknown in the art for use in pharmaceutical formulations, for example,for intravenous administration a carrier may be sodium chloride 0.9% ormixtures of normal saline with glucose or mannose. The choice of acarrier for use in a composition will depend upon the intended route ofadministration for the composition. The preparation of pharmaceuticallyacceptable carriers and formulations containing these materials isdescribed in, e.g., Remington's Pharmaceutical Sciences, 21st Edition,ed. University of the Sciences in Philadelphia, Lippincott, Williams &Wilkins, Philadelphia Pa., 2005, which is incorporated herein byreference in its entirety.

Formulations for Administration.

For therapeutic purposes, formulations for parenteral administration ofcompositions comprising BGP extracts can be in the form of aqueous ornon-aqueous isotonic sterile injection solutions or suspensions. Theterm parenteral, as used herein, includes intravenous, intravesical,intraperitoneal, subcutaneous, intramuscular, intralesional,intracranial, intrapulmonal, intracardial, intrasternal, and sublingualinjections, or infusion techniques.

Topical Administration.

Formulations for topical administration to the skin, wounds, burns, ormucous membranes include, for example, ointments, creams, gels andpastes comprising the composition in a pharmaceutical acceptablecarrier. The formulation of the composition for topical use includes thepreparation of oleaginous or water-soluble ointment bases, as is wellknown to those in the art. For example, these formulations may includevegetable oils, animal fats, and, for example, semisolid hydrocarbonsobtained from petroleum. Particular components used may include whiteointment, yellow ointment, cetyl esters wax, oleic acid, olive oil,paraffin, petrolatum, white petrolatum, spermaceti, starch glycerite,white wax, yellow wax, lanolin, anhydrous lanolin and glycerylmonostearate. Various water-soluble ointment bases may also be used,including glycol ethers and derivatives, polyethylene glycols, polyoxyl40 stearate and polysorbates.

Oral Administration.

Solid preparations for oral administration may include a tablet, a pill,a powder, a granule, a capsule, and the like. These solid preparationsmay be prepared by mixing at least one excipient, such as starch,calcium carbonate, sucrose, lactose, or gelatin, with nanoparticle asdisclosed herein. In addition to such a simple excipient, lubricants,such as magnesium stearate and talc, may be used. Liquid preparationscontaining BGP extracts for oral administration correspond to asuspension, a liquid for internal use, oil, syrup, and the like, and mayinclude several types of excipient, for example, a wetting agent, asweetener, an aroma, a preservative, and the like, in addition to simplediluents that are frequently used, such as water and liquid paraffin.

Parenteral administration. Injectable preparations, for example, sterileinjectable aqueous or oleaginous suspensions containing BGP extract canbe formulated according to the known art using suitable dispersing orwetting ingredients and suspending ingredients. The sterile injectablepreparation can also be a sterile injectable solution or suspension in anon-toxic parenterally acceptable diluent or solvent, for example, as asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat can be employed are water, Ringer's solution, and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil can be employed including synthetic mono- or diglycerides. Inaddition, fatty acids, such as oleic acid, find use in the preparationof injectables. Dimethyl acetamide, surfactants including ionic andnon-ionic detergents, polyethylene glycols can be used. Mixtures ofsolvents and wetting ingredients such as those discussed above are alsouseful.

Respiratory System Administration.

Administration to the respiratory system may be accomplished using adrug delivery device such as a nebulizer to administer a nanoparticlecomposition as disclosed herein, in an inhalable form. Nebulizersinclude soft mist inhalers, jet nebulizers, ultrasonic wave nebulizers,and nebulizers using vibrating mesh technology. A metered-dosage inhaleris another drug delivery device that delivers a selected or meteredamount of a medication, such as the nanoparticle compositions disclosedherein. Typically, this device produces and releases an aerosol ofmicrometer-sized particles that are inhaled. The aerosol will comprisinga BGP extract or component thereof as disclosed herein and may containexcipients or carriers.

Thus, preferably, particles comprising an extract are sized so as toprovide a uniform dosage or so they are absorbed in a particular part ofthe respiratory system. In some cases, the particles may be a dry powderin others as a mist or in a semiliquid form. Metered-dose inhalers andtheir various components, propellants, excipients and other elements aredescribed by and incorporated by reference to hypertext transferprotocol secure://en.wikipedia.org/wiki/Metered-dose_inhaler. Aninhalable composition may be formulated in the form of ahydrofluoroalkane inhaler or HFA (metered dose inhaler or MDI), drypowder inhaler (DPI), or as a nebulizer solution.

Dose.

The dose of a BGP extract as disclosed herein with respect to the humanor animal body may vary depending on patient's age, body weight, andgender, the form of administration, state of health, and severity ofdisease. The dose may be generally 0.01-100 mg/kg/day, preferably 0.1-20mg/kg/day, and more preferably 5-10 mg/kg/day. The composition may alsobe divisionally administered at predetermined intervals according toconverted or adjusted based on body surface area, e.g., to mg/m². Suchadjustments or conversions may be calculated using formulas known in theart; see DuBois D & DuBois E F. A formula to estimate the approximatesurface area if height and weight be known. ARCH INTERN MEDICINE. 1916;17:863-71; and Wang Y, Moss J, Thisted R. Predictors of body surfacearea. J CLIN ANESTH. 1992; 4(1):4-10, both of which are incorporated byreference.

An amount of BGP ethanol or oil extraction to be applied to a tissue orskin surface may be selected by one skilled in the medical orpharmaceutical arts. It may be based on the topical application of aparticular concentration of an ethanol or oil extract contained in atopically-applied composition, for example, application of a compositioncontaining a concentration of BGP ethanol or oil extract ranging from0.01, 0.1, 0.2, 0.5, 1, 2, 5, 10, 50 to 100 vol/vol. % of the extract.In other embodiments, the concentration of the extract is based on itsdry or residual content after removal of the solvent, such as removal ofethanol and water from an ethanol extract, or removal of oil from an oilextract. For example, a topical product may contain from 0.001, 0.002,0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 or >10 mg/kg or mg/Lof the dry or residual extract after solvent removal.

Nociceptive pain. Nociception is the sensory nervous system's process ofencoding noxious stimuli. In nociception, intense chemical (e.g.,cayenne powder), mechanical (e.g., cutting, crushing), or thermal (heatand cold) stimulation of sensory nerve cells called nociceptors producesa signal that travels along a chain of nerve fibers via the spinal cordto the brain. Nociception triggers a variety of physiological andbehavioral responses and usually results in a subjective experience, orperception, of pain in sentient beings. Potentially damaging mechanical,thermal, and chemical stimuli are detected by nerve endings callednociceptors, which are found in the skin, on internal surfaces such asthe periosteum, joint surfaces, and in some internal organs. Somenociceptors are unspecialized free nerve endings that have their cellbodies outside the spinal column in the dorsal root ganglia. Othernociceptors rely on specialized structures in the skin to transducenoxious information such as nociceptive schwann cells. Nociceptors arecategorized according to the axons which travel from the receptors tothe spinal cord or brain. After nerve injury it is possible for touchfibers that normally carry non-noxious stimuli to be perceived asnoxious.

Nociceptors have a certain threshold; that is, they require a minimumintensity of stimulation before they trigger a signal. Once thisthreshold is reached a signal is passed along the axon of the neuroninto the spinal cord.

Nociceptive threshold testing deliberately applies a noxious stimulus toa human or animal subject in order to study pain. In animals, thetechnique is often used to study the efficacy of analgesic drugs and toestablish dosing levels and period of effect. After establishing abaseline, the drug under test is given and the elevation in thresholdrecorded at specified time points. When the drug wears off, thethreshold should return to the baseline (pre-treatment) value.

Potentially damaging mechanical, thermal, and chemical stimuli aredetected by nerve endings called nociceptors, which are found in theskin, on internal surfaces such as the periosteum, joint surfaces, andin some internal organs. Some nociceptors are unspecialized free nerveendings that have their cell bodies outside the spinal column in thedorsal root ganglia. Other nociceptors rely on specialized structures inthe skin to transduce noxious information such as nociceptive schwanncells. Nociceptors are categorized according to the axons which travelfrom the receptors to the spinal cord or brain. After nerve injury it ispossible for touch fibers that normally carry non-noxious stimuli to beperceived as noxious.

Nociceptors have a certain threshold; that is, they require a minimumintensity of stimulation before they trigger a signal. Once thisthreshold is reached a signal is passed along the axon of the neuroninto the spinal cord. Nociceptive threshold testing deliberately appliesa noxious stimulus to a human or animal subject in order to study pain.In animals, the technique is often used to study the efficacy ofanalgesic drugs and to establish dosing levels and period of effect.

Pain.

The sensation of pain is associated with the activation of the receptorsin the primary afferent fibers, which is inclusive of the unmyelinatedC-fiber and myelinated Aa-fiber. Both nociceptors remain silent duringhomeostasis in the absence of pain and are activated when there is apotential of noxious stimulus. The perception of a series of sensoryevents is required for the brain in order to detect pain and produce aresponse towards the threat. There are generally three main stages inthe perception of pain. The first stage is pain sensitivity, followed bythe second stage where the signals are transmitted from the periphery tothe dorsal horn (DH), which is located in the spinal cord via theperipheral nervous system (PNS). Lastly, the third stage is to performthe transmission of the signals to the higher brain via the centralnervous system (CNS). Typically, there are two routes for signaltransmissions to be conducted: ascending and descending pathways. Thepathway that goes upward carrying sensory information from the body viathe spinal cord towards the brain is defined as the ascending pathway,whereas the nerves that goes downward from the brain to the reflexorgans via the spinal cord is known as the descending pathway.Nociceptive pain is the normal response to noxious (intense)stimulation.

C-Fiber Mediated Pain.

Group C nerve fibers are one of three classes of nerve fiber in thecentral nervous system (CNS) and peripheral nervous system (PNS). The Cgroup fibers are unmyelinated and have a small diameter and lowconduction velocity, whereas Groups A and B are myelinated. Group Cfibers include postganglionic fibers in the autonomic nervous system(ANS), and nerve fibers at the dorsal roots (IV fiber). These fiberscarry sensory information. Damage or injury to nerve fibers causesneuropathic pain. Capsaicin activates C fiber vanilloid receptors,giving chili peppers a hot sensation. Besides non-myelinated C fibers,pain may be afferently transmitted via thinly myelinated A-sigma, mediummyelinated A-beta, or thickly mediated A-alpha fibers.

NMDA Receptor Mediated Pain.

Receptors called “N-methyl-D-aspartate receptors” (NMDA receptors) havebeen shown to mediate chronic pain and visceral pain, including the painfrom distending a balloon in the colon. These receptors are expressed inthe spinal column and in peripheral tissues.

Nitric Oxide and Inflammatory Pain.

Nitric oxide (NO) is a signaling molecule that plays a key role in thepathogenesis of inflammation. It gives an anti-inflammatory effect undernormal physiological conditions. On the other hand, NO is considered asa pro-inflammatory mediator that induces inflammation due to overproduction in abnormal situations. NO is synthesized and released intothe endothelial cells by the help of NOSs that convert arginine intocitrulline producing NO in the process. Oxygen and NADPH are necessaryco-factors in such conversion. NO is believed to induce vasodilatationin cardiovascular system and furthermore, it involves in immuneresponses by cytokine-activated macrophages, which release NO in highconcentrations. In addition, NO is a potent neurotransmitter at theneuron synapses and contributes to the regulation of apoptosis. NO isinvolved in the pathogenesis of inflammatory disorders of the joint, gutand lungs.

Inflammatory pain refers to increased sensitivity due to theinflammatory response associated with tissue damage. Under thesesensitized conditions, an innocuous stimulus can be perceived aspainful—this is known as allodynia—and the pain evoked by a noxiousstimulus is exaggerated in both amplitude and duration—this is known ashyperalgesia. Inflammatory pain is a type of nociceptive pain thatresults from activation and sensitization of nociceptors by inflammatorymediators or increased sensitivity due to the inflammatory responseassociated with tissue damage.

Primary hyperalgesia is a direct consequence of peripheralsensitization, the process whereby inflammatory mediators and otherlocal factors—prostaglandin, bradykinin, cytokines (e.g, interleukin),histamine, serotonin, protons, potassium, ATP, glutamate, and soon—trigger intracellular signaling pathways in the peripheral terminalsof primary afferents, leading to changes in the function and/orexpression of receptor molecules and voltage-gated ion channels, whichultimately causes those afferents to become hyperresponsive tostimulation. Notably, peptidergic C fibers can release substance P andcalcitonin gene-related peptide (CGRP) from their peripheral terminals,causing neurogenic inflammation, thus forming a nasty positive feedbackloop. Through a phenomenon known as an axon reflex, in which spikesinitiated in one fiber branch propagate antidromically down neighboringfiber branches, neurogenic inflammation can extend throughout thereceptive field of a peptidergic C fiber. The hypersensitivity canspread even further through the effects of central sensitization, whichinvolves synaptic plasticity and other changes in the downstream centralcircuits. Synaptic plasticity and numerous other changes contribute tomaking the pain persistent but, generally speaking, inflammatory painpersists only as long as the inflammation. Reducing the inflammation istherefore a logical way to reduce the pain. Nonsteroidalanti-inflammatory drugs (NSAIDs), such as aspirin block the synthesis ofprostaglandins and thromboxanes via the enzyme cyclooxygenase. Furtherdescription of pain is described by and incorporate by reference to,hypertext transfer protocolsecure://www.sciencedirect.com/topics/medicine-and-dentistry/inflammatory-painhypertext transfer protocolsecure://www.sciencedirect.com/topics/medicine-and-dentistry/nociceptive-pain.Other types of pain include traumatic pain, arthritic pain, andpost-operative pain which are described by and incorporated by referenceto hypertext transfer protocolsecure://www.sciencedirect.com/topics/medicine-and-dentistry/inflammatory-pain.The compositions and methods disclosed herein may be applied totreatment of such types of pain.

Burns.

The treatment of burns depends on the depth, area and location of theburn. Burn depth is generally categorized as first, second or thirddegree. A first degree burn is superficial and has similarcharacteristics to a typical sun burn. The skin is red in color andsensation is intact. In fact, it is usually somewhat painful. Seconddegree burns look similar to the first degree burns; however, the damageis now severe enough to cause blistering of the skin and the pain isusually somewhat more intense. In third degree burns the damage hasprogressed to the point of skin death. The skin is white and withoutsensation. The methods and compositions disclosed herein may be used totreat first, second or third degree burns, for example, by theapplication of a composition or burn dressing containing BGP ethanoland/or oil extract. In some cases, treatment will comprise prophylacticadministration of a composition comprising BGP ethanol or BGP oilextract.

At the time of burn injury, tissue damage is the primary mechanism ofpain. Stimulation of local nociceptors transmits an impulse via Ad and Cfibers to the dorsal horn of the spinal cord. Peripheral sensory nervesand descending influences from cortical areas can modulate the magnitudeof the pain impulse. Ultimately, conscious perception of pain isregulated by areas of the brain often named the “pain matrix” which isthought to involve a network of higher cortical areas and the thalamus.The conscious perception of pain is affected not only by the burn wounditself but also by context, cognition, pharmacologics, mood and otherpredisposing factors. Burn pain may also vary and fluctuate widely overthe span of recovery. Therefore, the successful treatment of burn painshould involve a multi-modal approach tailored to the patient andscenario. These and other features of burns and burn pain are describedby and incorporated by reference to Griggs, C. et al., CLIN PLAST SURG.2017 July; 44(3): 535-540. Prevention or immediate treatment of earlyevents in nociception of burn and other types of nociceptive pain mayreduce subsequent perception of other types of pain, such asinflammatory pain.

The mechanisms underlying burn-induced neuropathic pain have not beenstudied in detail. Acute pain arises from a range of noxious stimuliincluding heat, cold, mechanical stimuli, and chemicals. In the case ofburn-induced pain, damage to peripheral sensory neurons and inflammatoryprocesses initiated by the injury exacerbate this acute response andtransform burn pain into a complex symptom comprising multiplecomponents, including ongoing background pain and procedural pain duringsurgical interventions and dressing changes. In addition, bum-inducedpain can persist beyond the initial injury and develop into chronic painwith neuropathic features. However, the long list of the biological adpharmacological properties could make propolis a good analgesic agentfor different type of pains including burn pain. Biochemical andclinical features of burn pain are described by, and incorporated byreference to, Morgan, et al, Burn Pain: A Systematic and Critical Reviewof Epidemiology, Pathophysiology, and Treatment, PAIN MEDICINE, 2017,19(4). 708-734, In preferred embodiments, the compositions and methodsdisclosed herein are used to treat one or more features or symptoms ofburn pain.

The methods and compositions disclosed herein may be used to interruptearly events in nociception of burn pain as well as subsequently reducethe development or perception of inflammatory pain. A burn results incomplicated chemical and immunological inflammatory response that can berestricted to the site of injury or involve systemic inflammatoryprocesses. Activation of peripheral sensory nerve terminals after burninjuries as well as sustained nociceptive input post-injury can occurthrough a range of stimuli, including the initial thermal insult,vascular damage, hypoxia from ischemic events. DAMP receptor activation,and local release of pro-inflammatory and algogenic mediators andtransmitters such as bradykinin, histamine. eicosanoids,platelet-activating factor (PAF), nerve growth factor (NGF), SubstanceP, and CGRP, trigger a syndrome associated with significant morbidityand mortality. Further description of these phenomena are described by,and incorporated by reference to Morgan, et al., supra. The compositionsand methods disclosed herein may interrupt or attenuate one or more ofthese phenomena.

Assays for evaluating or measuring nociceptive pain include thosedescribed in the Examples as well as those described by, andincorporated by reference to, hypertext transfer protocolsecure://en.wikipedia.org/wiki/Nociception_assay (last accessed Jun. 1,2021). A nociception assay (nocioception or nocioperception assay)evaluates the ability of an animal, usually a rodent, to detect anoxious stimulus such as the feeling of pain, caused by stimulation ofnociceptors. These assays measure the existence of pain throughbehaviors such as withdrawal, licking, immobility, and vocalization. Inanimals (rats), signs of pain are measured by quantifiable behavioralcomponents; examples of such behaviors include paw withdrawal latency inreaction to thermal noxious stimulation (heat, cold), frequency andthreshold of paw withdrawal in reaction to mechanical stimulation,degree of self-mutilation (autotomy), or spontaneous lifting and lickingof paws. These reactions are interpreted as equivalent to mechanical orthermal hyperalgesia or allodynia or spontaneous pain in humans.

Example

Methodology. Experimental Animals.

Wistar male albino rats (250-300 g) were obtained from the animal houseof the Imam Abdulrahman Bin Faisal University. Animals were maintainedwith free access to water and food at controlled room temperature 25-29°C., 12-h light and 12 h darkness cycles”; Al-Hariri, M. T.; et al.,Protective effect and potential mechanisms of propolis onstreptozotocin-induced diabetic rats. J. TAIBAH UNIV. MED SCI. 2016, 11,7-12, incorporated herein by reference in its entirety. The experimentwas performed after approval of the protocols by UniversityInstitutional Review Board Committee (IRB 2015-03-114) and was carriedout in accordance with the current guidelines for the care of laboratoryanimals.

Rats were distributed into three random groups (n=6);

Group I is a control group that received normal saline intraperitoneally(i.p.);

Group II was treated with high flavonoids Brazilian green propolis (55%)ethanol extracts (Uniflora) 50 mg/kg (P50) i.p. and

Group III was treated with 100 mg/kg (P100) i.p.

Animal groups were treated 60 min before testing; Deuis, J. R. et al.,Methods used to evaluate pain behaviors in rodents. FRONT. MOL.NEUROSCI. 2017, 10, 284, incorporated herein by reference in itsentirety.

Animal Models of Pain include the tests and assays which are describedbelow.

Thermal Hyperalgesia (Infrared) Test.

Thermal hyperalgesia was assessed using a planter analgesic meter (UgoBasile, Gemonio, Italy). Rats were placed individually in clear plasticcages and allowed to acclimate for 20 min before testing. The center ofa focused beam of infrared heat was applied to the plantar surface ofthe hind paws. Both timer and bulb were immediately turned off by rat'spaw withdrawal or on reaching a predetermined cut-out (usually 22 s) toprevent tissue damage. The reaction time was recorded when the ratslicked their hind paws and jumped at several intervals of 30, 60 and 90min post propolis administration. The withdrawal latency of hind pawswas measured to the nearest 0.1 s. See Deuis, J. R. & Vetter, I. Thethermal probe test: A novel behavioral assay to quantify thermal pawwithdrawal thresholds in mice. TEMPERATURE 2016, 3, 199-207,incorporated herein by reference in its entirety.

Formalin Test.

Rats were treated with different doses (P50 and P100) of propolis i.p.After sixty minutes, 50 μL of 5% formalin was injected subcutaneouslyinto the dorsal surface of the right hind paw using a microsyringe witha 27-gauge needle. Immediately after formalin injection, the animalswere placed individually in acrylic observation chambers (320 cm×40 cm).Mirrors were arranged at angles to allow clear observation of the pawsof the animals. Each rat in every group was observed simultaneously from0 to 60 min following formalin injection.

The nociceptive response was identified by counting the number offlinches of the injected paw. The number of flinches was counted for theperiods of 0-5 min (neurogenic phase) and 10-60 min (Inflammatoryphase). See Hong, J.-S.; et al. Antinociceptive effect of chrysin indiabetic neuropathy and formalin-induced pain models. ANIM. CELLS SYST.2020, 24, 1-8, incorporated herein by reference in its entirety.

Abdominal Writhing test. Abdominal writhe (contraction) was induced byi.p. injection of 1 mL of a solution of 2% acetic acid using a 25-gaugeinjection needle. Five minutes after the administration of acetic acid,the frequencies of abdominal writhing were recorded for sixty minutesand, the latent period, i.e., beginning of the first abdominal writheafter injection, was also observed. See Bahamonde et al.,Antinociceptive and anti-inflammatory activities of an aqueous extractof Chiliotrichum diffusum. REVISTA BRASILIERA DE FARMACOGNOSIA. 2013,23(4), 699-705; incorporated by reference.

Statistical Analysis.

Statistical analysis was performed using the Statistical Package for theSocial Sciences (IBM SPSS V 21). The normality of data were tested bythe Shapiro-Wilk. The number of flinches of all phases of response werecalculated for each rat. Data with homogeneity of variance were analyzedusing the two-way analysis of variance (ANOVA), and multiple post hoccomparisons were performed using the Tukey's test. p-values <0.05 wereconsidered to be statistically significant. Data were expressed as meanf standard error of mean (SEM).

Inhibitory Effect of Propolis on Thermal Stimuli-Induced Nociception inthe Infrared Test.

Administration of Brazilian green propolis alcohol extract at doses 50and 100 mg/kg, i.p. increased the latency time to the nociceptiveresponse in the infrared test. Significant differences (p<0.05) betweenthe mean thermal threshold of P50 and P100 than the control, clearlyindicating the analgesic property of the alcohol Brazilian greenpropolis alcohol extract as presented in FIG. 1 .

Inhibitory Effects of Propolis on Formalin-Induced InflammatoryNociception.

FIG. 2 shows the effect of the Brazilian green propolis alcohol extracton formalin-induced pain during the acute phase in rats. Both propolisgroups (P50 and P100) displayed a significant (p<0.05) reduction informalin-induced nociceptive behavior monitored by the mean number offlinches across all time points of the neurogenic phase (0-5 min) thanthe control. Similarly, a significant (p<0.05) pain inhibition in bothgroups (P50 and P100) was observed in the inflammatory phase (10-60 min)after induction of the noxious stimulus, than the control.

Inhibitory Effect of Propolis on Acetic Acid-Induced Writhing Response.

The effect of the Brazilian green propolis ethanol extract on theantinociceptive activity as determined by the acetic acid-inducedabdominal writhing test is shown in FIG. 3 . Both the 50 and 100 mg/kg,i.p. does of the BGO ethanol extract significantly (p<0.05) decreasedacetic acid-induced writhing responses compare to responses in thecontrol group.

The data presented above show that administration of BGP ethanol extractsignificantly reduced nociceptive responses as determined severalwell-established in vivo nociception assays which measure different painstimuli including both chemical and thermal pain stimuli. Administrationof Brazilian green propolis alcohol extract reduced the number offlinching response evoked by formalin injection during both phases. Theearly phase started at the time of injection and lasted for about fiveminutes and the late phase, which started at 10 minutes post-injectionand lasted for about 60 minutes. The early phase response results fromimmediate stimulation of primary afferent fibers (C-fiber activation dueto the peripheral stimulus) in the paw that reflects the centrallymediated pain. The late phase response may result from an inflammatoryreaction in the peripheral tissue, combined with the activation of widedynamic range neurons (activation of N-methyl-D-aspartate (NMDA) andnon-NMDA receptors and NO cascade) in the dorsal horn of the spinalcord. Further description of these responses and pain stimuli areincorporated by reference to Hunskaar, S. & Hole, K. The formalin testin mice: Dissociation between inflammatory and non-inflammatory pain.PAIN 1987, 30, 103-114; and Puig, S.; Sorkin, L. Formalin-evokedactivity in identified primary afferent fibers: Systemic lidocainesuppresses phase-2 activity. P AIN 1996, 64, 345-355.

The inventors considered that Brazilian green ethanol extract possessesa biphasic analgesic effect. As shown above, Brazilian green propolisethanol extract causes a significant inhibition of acetic acid-inducedpain that reduced the number of abdominal contraction and stretching ofhind limbs. While not being bound to any theory or technicalexplanation, this acetic acid induced nociceptive response may involveboth the synthesis of inflammatory mediators which reduce the thresholdof nociception and the direct stimulation of the nociceptive afferentfibers due to the reduction of pH. Moreover, the antinociceptive effectof Brazilian green propolis alcohol extract against acetic acid-inducedwrithing may occur via local peritoneal receptors or by inhibition ofprostaglandin synthesis or action.

As shown and disclosed herein, the inventors have found that a BGPethanol extract exhibits an important analgesic effects includinginhibition of pain threshold. These results demonstrate that an extractof a natural product, BGP, may be used in the treatment of severalpainful and/or inflammatory diseases or conditions, especially in themanagement of nociceptive pain associated with thermal injury.

1. A method for treating nociceptive pain from a burn comprising:administering to a subject in need of treatment for nociceptive painfrom a bum afferently transmitted via non-myelinated C fibers acomposition comprising an ethanol extract of Brazilian green propolis(“BGP”).
 2. The method of claim 1, wherein the pain from the burn isnon-inflammatory pain.
 3. The method of claim 1, wherein the nociceptivepain is an early phase response mediated by myelinated A-sigma, A-beta,or A-alpha fibers.
 4. The method of claim 1, wherein the nociceptivepain further comprises late phase response resulting from aninflammatory response in combination with activation ofN-methyl-d-aspartate (NMDA) and non-NMDA receptors and NO cascade). 5.The method of claim 1, wherein the ethanol extract of BGP is produced byextracting BGP with an aqueous solution of at least 70% ethanol.
 6. Themethod of claim 1, wherein the composition further comprises an oilextract of BGP.
 7. The method of claim 1, wherein the compositionfurther comprises a vegetable oil extract of BGP.
 8. The method of claim1, wherein the composition further comprises a vegetable oil extract ofBGP that has been emulsified with the ethanol extract of BGP.
 9. Themethod of claim 1, where the composition further comprises a NSAID or anopioid.
 10. The method of claim 1, wherein the composition furthercomprises a sunblock or sunscreen and is topically administered to thesubject.
 11. The method of claim 1, wherein the composition is topicallyadministered to the subject.
 12. The method of claim 1, wherein thecomposition is administered to the subject prior to a burn.
 13. Themethod of claim 1, further comprising exposing at least a portion of theburn of the subject in need of treatment to UV light at an intensity of5-50 mW/cm² for up to ½ hour ½ hour or less prior to administering thecomposition comprising the BGP ethanol extract to the burn wherein theUV light is in a wavelength range of 280-400 nm.
 14. A gel or spraycomprising an ethanol extract of BGP and an excipient suitable forapplication to a burn.
 15. The gel or spray of claim 14, furthercomprising an oil extract of BGP.
 16. The gel or spray of claim 14,further comprising an NSAID or opioid.
 17. The gel or spray of claim 14that comprises a hydrogel.
 18. The gel or spray of claim 14 thatcomprises a sunscreen or sunblock.
 19. A burn dressing comprising anethanol extract of BGP.
 20. The burn dressing of claim 19, furthercomprising an oil extract of BGP.